1. Field of the Invention
The present invention relates to a population of insulin producing cells differentiated from non-insulin producing cells by contacting the non-insulin producing cells with Glucagon-like peptide-1 (“GLP-1”), exendin-4, or related peptides. The present invention also relates to the methods for obtaining the insulin producing cells and therapeutic uses in the treatment of diabetes mellitus.
2. Background Art
The mammalian pancreas is composed of two distinct types of glandular tissue, the exocrine cells that secrete digestive enzymes into the intestine and the endocrine cells that secrete hormones into the blood stream. Endocrine cells were traditionally believed to develop from the neural crest whereas the exocrine cells were believed to develop from the endoderm. More recent work suggests that these two cell types can come from common endodermal precursor cells located along the epithelial lining of the ducts (Teitelman, 1996). It should be noted that the endocrine cells are terminally differentiated and do not divide to make new endocrine cells. Pancreatic endodermal precursor cells are the only cells thought to produce new pancreatic endocrine cells.
The pancreas consists of ducts, which carry the exocrine enzymes (amylase and lipase) to the intestine; acinar cells, which produce the exocrine enzymes; and islets of Langerhans, which contain the endocrine cells that produce and secrete insulin, amylin, and glucagon. These hormones help to maintain normal blood glucose levels within a remarkably narrow range.
Among the islet cells are beta cells which produce and secrete insulin. Insulin production and secretion by the beta cells is controlled by blood glucose levels. Insulin release increases as blood glucose levels increase. Insulin promotes the uptake of glucose by target tissues and, thus, prevents hyperglycemia by shuttling glucose into tissues for storage.
Beta cell dysfunction and the concomitant decrease in insulin production can result in diabetes mellitus. In Type 1 diabetes, the beta cells are completely destroyed by the immune system, resulting in an absence of insulin producing cells (Physician's Guide to Insulin Dependent [Type I] Diabetes Mellitus: Diagnosis and Treatment, American Diabetes Association, 1988). In Type 2 diabetes, the beta cells become progressively less efficient as the target tissues become resistant to the effects of insulin on glucose uptake. Type 2 diabetes is a progressive disease and beta cell function continues to deteriorate despite on-going treatment with any presently available agent (UK Prospective Study Group, 1995). Thus, beta cells are absent in people with Type 1 diabetes and are functionally impaired in people with Type 2 diabetes.
Beta cell dysfunction currently is treated in several different ways. In the treatment of Type 1 diabetes or the late stages of Type 2 diabetes, insulin replacement therapy is used. Insulin therapy, although life-saving, does not restore normoglycemia, even when continuous infusions or multiple injections are used in complex regimes. For example, postprandial levels of glucose continue to be excessively high in individuals on insulin replacement therapy. Thus, insulin therapy must be delivered by multiple daily injections or continuous infusion and the effects must be carefully monitored to avoid hyperglycemia, hypoglycemia, metabolic acidosis, and ketosis.
Replacement of beta cells can be achieved with pancreatic transplants. (Scharp et al., 1991; Warnock et al., 1991). Such transplants, however, require finding a matching donor, surgical procedures for implanting the harvested tissue, and graft acceptance. After transplantation in a person with Type 1 diabetes, on-going immunosuppression therapy is required because cell surface antigens on the beta cells are recognized and attacked by the same processes that destroyed the beta cells originally. Immunosuppressive drugs, such as cyclosporin A, however, have numerous side-effects, including the increase in potential for infection. Transplantation, therefore, can result in numerous complications.
People with Type 2 diabetes are generally treated with drugs that stimulate insulin production and secretion from the beta cells. A major disadvantage of these drugs, however, is that insulin production and secretion is promoted regardless of the level of blood glucose. Thus, food intake must be balanced against the promotion of insulin production and secretion to avoid hypoglycemia or hyperglycemia.
In recent years several new agents have become available to treat Type 2 diabetes. These include metformin, acarbose and troglitazone (see Bressler and Johnson, 1997). However, the drop in hemoglobin A1c obtained by these newer agents is less than adequate (Ghazzi et al., 1997), suggesting that they will not improve the long-term control of diabetes mellitus.
Most recently, glucagon-like peptide-1 (GLP-1), a hormone normally secreted by neuroendocrine cells of the gut in response to food, has been suggested as a new treatment for Type 2 diabetes (Gutniak et al., 1992; Nauck et al., J. Clin. Invest., 1993). It increases insulin release by the beta cells even in subjects with long-standing Type 2 diabetes (Nauck et al., Diabetologia, 1993). GLP-1 treatment has an advantage over insulin therapy because GLP-1 stimulates endogenous insulin secretion, which turns off when blood glucose levels drop (Nauck et al., Diabetologia, 1993; Elahi et al., 1994), when blood glucose levels are high. GLP-1 promotes euglycemia by increasing insulin release and synthesis, inhibiting glucagon release, and decreasing gastric emptying (Nauck et al., Diabetologia, 1993; Elahi et al., 1994; Wills et al., 1996; Nathan et al., 1992; De Ore et al., 1997). GLP-1 also induces an increase in hexokinase messenger RNA levels (Wang et al., Endocrinology 1995; Wang et al., 1996). GLP-1 is known to have a potent insulin-secreting effect on beta cells (Thorens and Waeber, 1993; Orskov, 1992) and to increase insulin biosynthesis and proinsulin gene expression when added to insulin-secreting cell lines for 24 hours (Drucker et al., 1987; Fehmann and Habener, 1992). In studies using RIN 1046-38 cells, twenty-four hour treatment with GLP-1 increased glucose responsiveness even after the GLP-1 had been removed for an hour and after several washings of the cells (Montrose-Rafizadeh et al., 1994). Thus, GLP-1 is an insulin releasing agent and an insulinotropic agent (i.e., an agent that increases insulin synthesis) known to have a prolonged effect on beta cells. GLP-1 is a product of posttranlational modification of proglucagon. The sequences of GLP-1 and its active fragments GLP-1 (7–37) and GLP-1 (7–36) amide are known in the art (Fehmann et al., 1995).
GLP-1 receptors have been shown to be present in the gut and in the pancreatic islets (Id.). The receptors belong to a family of G-protein-linked receptors that includes glucagon, secretin, and vasoactive intestinal peptide receptors. After binding of GLP-1 to its receptor there is a rise in cAMP in beta cells of the islets of Langerhans (Widmann et al., 1996), indicating that the receptor is coupled to the adenyl cyclase system by a stimulator G-protein. In peripheral tissues, such as liver, fat and skeletal muscle, however, no increase in cAMP with GLP-1 is seen, suggesting that GLP-1 acts through a different system on peripheral tissues (Valverde and Villanueva-Penacarrillo, 1996).
Exendin-4 is a peptide produced in the salivary glands of the Gila Monster lizard (Goke et al., 1993). The amino acid sequence for Exendin-4 is known in the art (Fehmann et al. 1995). Although it is the product of a uniquely non-mammalian gene and appears to be expressed only in the salivary gland (Chen and Drucker, 1997), Exendin-4 shares a 52% amino acid sequence homology with GLP-1 and in mammals interacts with the GLP-1 receptor (Goke et al., 1993; Thorens et al., 1993). In vitro, Exendin-4 has been shown to promote insulin secretion by insulin producing cells and, given in equimolar quantities, is more potent than GLP-1 at causing insulin release from insulin producing cells.
In vivo studies using GLP-1 have been limited to the use of single or repeated bolus injections or short-term infusions of GLP-1 and subsequent evaluation of the insulin secreting effects. In one such study, infusions of GLP-1 for two hours were tested in patients with Type 1 diabetes for the ability of GLP-1 to promote glucose uptake in muscle and release of glucose from the liver (Gutniak et al., 1992). Therapeutic uses of GLP-1 for increasing the release of insulin have been considered for Type 2 diabetes, but not for Type 1 diabetes, since Type 1 diabetes is marked by an absence of beta cells, the known target cell for GLP-1. Furthermore, GLP-1 has known limitations as a therapeutic agent in the treatment of diabetes because it has a short biological half-life (De Ore et al., 1997), even when given by a bolus subcutaneously (Ritzel et al., 1995). Exendin-4 has not been used previously in in vivo studies. Thus, studies to date have never suggested that either GLP-1 or Exendin-4 is therapeutically effective on pancreatic function in people with Type 1 diabetes or that there are GLP-1 or Exendin-4 target cells in the pancreas other than the beta cells.